U.S. patent application number 09/852512 was filed with the patent office on 2002-01-24 for methods for identifying modulators of neuronal growth.
Invention is credited to Miller, Freda D., Vaillant, Andrew.
Application Number | 20020009713 09/852512 |
Document ID | / |
Family ID | 26898710 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009713 |
Kind Code |
A1 |
Miller, Freda D. ; et
al. |
January 24, 2002 |
Methods for identifying modulators of neuronal growth
Abstract
The invention features methods identifying compounds that
modulate neuronal growth. The invention also features methods of
modulating neuronal growth by modulating the p75.sup.NTR or
MEK/MAPK pathways, and methods of identifying compounds that do the
same.
Inventors: |
Miller, Freda D.; (Montreal,
CA) ; Vaillant, Andrew; (Montreal, CA) |
Correspondence
Address: |
CLARK & ELBING LLP
176 FEDERAL STREET
BOSTON
MA
02110-2214
US
|
Family ID: |
26898710 |
Appl. No.: |
09/852512 |
Filed: |
May 10, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60203560 |
May 11, 2000 |
|
|
|
Current U.S.
Class: |
435/4 ; 435/368;
435/6.16 |
Current CPC
Class: |
C12N 9/1205 20130101;
C12N 5/0619 20130101; G01N 33/5035 20130101; G01N 33/5058 20130101;
A01K 67/0275 20130101; C12N 2830/008 20130101; C12N 2503/02
20130101; G01N 33/5026 20130101; A01K 2267/0356 20130101; C12Q
1/6897 20130101; C12N 2501/13 20130101; G01N 33/5008 20130101 |
Class at
Publication: |
435/4 ; 435/6;
435/368 |
International
Class: |
C12Q 001/00; C12Q
001/68; C12N 005/08 |
Claims
What is claimed is:
1. A method for identifying a compound that increases or decreases
neuritic growth, said method comprising the steps of: (a)
contacting a culture comprising neurons with a test compound; and
(b) measuring the amount of a protein or RNA that is preferentially
found in the neurites of said neurons, wherein an increase in the
amount of said protein or RNA relative to the amount of said
protein or RNA in said culture not contacted with said test
compound identifies said test compound as a compound that increases
neuritic growth and a decrease in the amount of said protein or RNA
relative to the amount of said protein or RNA in said culture not
contacted with said test compound identifies said test compound as
a compound that decreases neuritic growth.
2. The method of claim 1, wherein said protein is selected from the
group consisting of alpha-tubulin, beta-tubulin, GAP-43,
neurofilament light chain, neurofilament medium chain,
neurofilament heavy chain, microtubule-associated protein (MAP) 1B,
MAP4, MAP2C, and MAP2D.
3. The method of claim 1, wherein said neurons comprise sympathetic
neurons, cholinergic neurons, sensory neurons, cortical neurons,
motor neurons, and/or dorsal root ganglion neurons.
4. The method of claim 1, wherein said neurons comprise primary
neurons, neurons derived from a cell line, and/or neurons derived
from a stem cell.
5. The method of claim 1, wherein the test compound is selected
from the group consisting of a peptide or protein, a carbohydrate,
a lipid, a nucleic acid molecule, a natural organic molecule, and a
synthetically derived organic molecule.
6. A method for identifying a compound that increases or decreases
dendritic growth, said method comprising the steps of: (a)
contacting a culture comprising neurons with a test compound; and
(b) measuring the amount of a protein or RNA that is preferentially
found in the dendrites of said neurons, wherein an increase in the
amount of said protein or RNA relative to the amount of said
protein or RNA in said culture not contacted with said test
compound identifies said test compound as a compound that increases
dendritic growth and a decrease in the amount of said protein or
RNA relative to the amount of said protein or RNA in said culture
not contacted with said test compound identifies said test compound
as a compound that decreases dendritic growth.
7. The method of claim 6, wherein said protein is selected from the
group consisting of MAP2A, MAP2B, dendrin, type II
calcium-calmodulin-dependent protein (CamIIK), metabotropic
glutamate receptor (mGluR) 1A, mGluR2, and type 1A serotonin
receptor.
8. The method of claim 6, wherein said RNA is selected from the
group consisting of MAP2A RNA, MAP2B RNA, dendrin RNA, and CamIIK
RNA.
9. A method for identifying a compound that increases or decreases
axonal growth, said method comprising the steps of: (a) contacting
a culture comprising neurons with a test compound; and (b)
measuring the amount of a protein or RNA that is preferentially
found in the axons of said neurons, wherein an increase in the
amount of said protein or RNA relative to the amount of said
protein or RNA in said culture not contacted with said test
compound identifies said test compound as a compound that increases
axonal growth and a decrease in the amount of said protein or RNA
relative to the amount of said protein or RNA in said culture not
contacted with said test compound identifies said test compound as
a compound that decreases axonal growth.
10. The method of claim 9, wherein said protein is selected from
the group consisting of phosphorylated MAP1B, phosphorylated
neurofilament heavy chain, and type 1B serotonin receptor.
11. A method of identifying a compound that increases or decreases
neuronal growth, said method comprising the steps of: (a)
contacting a culture comprising neurons with NGF at levels
sufficient to support cell survival; (b) contacting said culture
with a neuronal growth-inhibiting compound; (c) contacting said
culture with a test compound; and (d) measuring the amount of a
protein or RNA that is preferentially found in the axons,
dendrites, or neurites of said neurons, wherein an increase in the
amount of said protein or RNA relative to the amount of said
protein or RNA in said culture not contacted with said test
compound identifies said test compound as a compound that increases
neuronal growth and a decrease in the amount of said protein or RNA
relative to the amount of said protein or RNA in said culture not
contacted with said test compound identifies said test compound as
a compound that decreases neuronal growth.
12. The method of claim 11, wherein said neuronal growth-inhibiting
compound is BDNF or an inhibitor of MEK signaling.
13. The method of claim 12, wherein said inhibitor of MEK signaling
is a dominant-negative MEK.
14. The method of claim 12, wherein said inhibitor of MEK signaling
is U0126 or PD98059.
15. A method of identifying a compound that increases or decreases
neuronal growth, said method comprising the steps of: (a)
inhibiting MEK signaling in a culture comprising neurons; (b)
contacting said culture with a test compound; and (c) measuring the
amount of a protein or RNA that is preferentially found in the
axons, dendrites, or neurites of said neurons, wherein an increase
in the amount of said protein or RNA relative to the amount of said
protein or RNA in said culture not contacted with said test
compound identifies said test compound as a compound that increases
neuronal growth and a decrease in the amount of said protein or RNA
relative to the amount of said protein or RNA in said culture not
contacted with said test compound identifies said test compound as
a compound that decreases neuronal growth.
16. The method of claim 15, wherein said MEK signaling is inhibited
by contacting said neuron with an inhibitor of MEK signaling.
17. The method of claim 16, wherein said inhibitor of MEK signaling
is a dominant-negative MEK.
18. The method of claim 16, wherein said inhibitor of MEK signaling
is U0126 or PD98059.
19. A method of identifying a compound that increases neuronal
growth, said method comprising: (a) activating p75.sup.NTR
signaling in a culture comprising neurons; (b) contacting said
culture with a test compound; and (c) measuring the amount of a
protein or RNA that is preferentially found in the axons,
dendrites, or neurites of said neurons, wherein an increase in the
amount of said protein or RNA relative to the amount of said
protein or RNA in said culture not contacted with said test
compound identifies said test compound as a compound that increases
neuronal growth and a decrease in the amount of said protein or RNA
relative to the amount of said protein or RNA in said culture not
contacted with said test compound identifies said test compound as
a compound that decreases neuronal growth.
20. The method of claim 19, wherein said p75.sup.NTR signaling is
activated by introducing ceramide into said neuron.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit from U.S. Provisional
Application Serial No. 60/203,560, filed May 11, 2000 (now
pending), which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The invention relates to neuronal growth.
[0003] The development of strategies to promote repair of the
degenerating or traumatized nervous system is a major ongoing
therapeutic challenge. One approach to the problem of nerve trauma
and regeneration is the development of relevant therapeutics that
can promote the survival or repair of traumatized neurons. One of
the fundamental technological problems associated with the
identification of such therapies, however, is the lack of suitable
high throughput screens for compounds effective on primary neurons.
This lack of suitable screens derives from two major
considerations. First, recent findings indicate that primary
neurons differ significantly in their survival and growth signaling
pathways from any of the transformed or immortalized cell lines
that are currently available, making screens using cell lines
unreliable. Second, postmitotic neurons are (i) only available in
relatively small amounts; (ii) difficult to genetically manipulate;
and (iii) difficult to culture as a purified cell population.
[0004] Neurons within the peripheral nervous system, including
those affected by peripheral neuropathy, have the capacity for
axonal growth and regeneration. Moreover, under certain conditions,
peripheral neurons are capable of axonal growth and regeneration
following axonal injury. It is not well understood what regulates
such axonal growth and regeneration. It would be desirable to
stimulate neuronal growth for the treatment of a neurodegenerative
disease or a neurotrauma. To this end, it would also be desirable
to more fully understand the signals that regulate neuronal
growth.
SUMMARY OF THE INVENTION
[0005] The invention features methods for identifying compounds for
the modulation of neuronal cell growth, including regeneration. The
invention also features a method and reagents for
modulatingneuronal growth. The method is based upon our discovery
that p75.sup.NTR and Trk-mediated MEK/MAPK signaling modulate
neuronal growth but not survival.
[0006] Accordingly, in a first aspect, the invention features a
method for identifying a compound that increases or decreases
neuronal growth. The method includes the steps of: (a) contacting a
culture that includes neurons with a test compound; and (b)
measuring the amount of a protein or RNA that is preferentially
found in the axons, dendrites, or neurites of the neurons. An
increase in the amount of the protein or RNA relative to the amount
of the protein or RNA in a culture not contacted with the test
compound identifies the test compound as a compound that increases
neuronal growth. Conversely, a decrease in the amount of the
protein or RNA relative to the amount of the protein or RNA in a
culture not contacted with the test compound identifies the test
compound as a compound that decreases neuronal growth.
[0007] Exemplary proteins that are preferentially found in neurites
include alpha-tubulin, beta-tubulin, GAP-43, neurofilament light
chain, neurofilament medium chain, neurofilament heavy chain,
microtubule-associated protein (MAP) 1B, MAP4, MAP2C, and MAP2D.
Proteins preferentially found in dendrites include, for example,
MAP2A, MAP2B, dendrin, type II calcium-calmodulin-dependent protein
(CamIIK), metabotropic glutamate receptor (mGluR) 1A, mGluR2, and
type 1A serotonin receptor (5-HT1a), while RNAs preferentially
found in dendrites include MAP2A RNA, MAP2B RNA, dendrin RNA, and
CamIIK RNA. Examples of proteins preferentially found in axons
include phosphorylated MAP1B, phosphorylated neurofilament heavy
chain, and type 1B serotonin receptor.
[0008] In another aspect, the invention features a method of
identifying a compound that increases neuronal growth, the method
including: (a) contacting a neuron with a neurotrophin at levels
sufficient to support cell survival; (b) contacting the neuron with
a neuronal growth-inhibiting compound; (c) contacting the neuron
with a test compound; and (d) assaying the level of neuronal growth
in the neuron, wherein an increase in neuronal growth relative to a
neuron contacted with the NGF and the neuronal growth-inhibiting
compound, but not contacted with the test compound, identifies the
test compound as a compound that increases neuronal growth.
[0009] It is desirable that the foregoing method be quantifiable.
In one example, the level of neuronal growth is assayed by
measuring the amount of total protein in a culture or by measuring
the level of a protein or mRNA that is preferentially found in
dendrites, axons, or both (i.e., neurites).
[0010] The neurotrophin can be, for example, NGF, BDNF, NT-3, or
NT-4/5. The neuronal growth-inhibiting compound can be, for
example, an inhibitor of MEK signaling. Such inhibitors are known
in the art (e.g., PD98059 (Pang et al., J. Biol. Chem.
270:13585-13588,1995); U0126 (Duncia et al., Bioorg. Med. Chem.
Lett. 8:2839-2844, 1998)). The neuronal growth-inhibiting compound
can also be an activator of p75.sup.NTR signaling. If the neuron
does not have TrkB receptors, the growth-inhibiting compound can be
BDNF or a BDNF analog.
[0011] In a related aspect, the invention features another method
of identifying a compound that increases neuronal growth. This
method includes (a) inhibiting MEK signaling in a neuron; (b)
contacting the neuron with a test compound; and (c) assaying the
level of neuronal growth in the neuron, wherein an increase in
neuronal growth relative to a neuron in which MEK signaling is
inhibited but which is not contacted with the test compound
identifies the test compound as a compound that increases neuronal
growth.
[0012] MEK signaling can be inhibited, for example, by expression
of a dominant-negative MEK in the neuron, or by contacting the
neuron with an inhibitor of MEK signaling (e.g., U0126 or PD98059).
It is desirable that MEK signaling is inhibited by at least 25%, by
at least 50%, or even by at least 75%.
[0013] In a related aspect, the invention features another method
of identifying a compound that increases neuronal growth, the
method including (a) contacting a neuron with a test compound; and
(b) assaying the level of MEK signaling in the neuron, wherein an
increase in MEK signaling relative to a neuron not contacted with
the test compound identifies the test compound as a compound that
increases neuronal growth.
[0014] In still another aspect, the invention features another
method of identifying a compound that increases neuronal growth.
This method includes (a) activating p75.sup.NTR signaling in a
neuron; (b) contacting the neuron with a test compound; and (c)
assaying the level of neuronal growth in the neuron, wherein an
increase in neuronal growth relative to a neuron in which
p75.sup.NTR signaling is activated but which is not contacted with
the test compound identifies the test compound as a compound that
increases neuronal growth.
[0015] p75.sup.NTR signaling can be activated, for example, by
introducing ceramide into the neuron. Preferably, p75.sup.NTR
signaling is activated by at least 25%, more preferably by at least
50%, and most preferably by at least 75%.
[0016] In a related aspect, the invention features a method of
identifying a compound that increases neuronal growth, the method
including (a) contacting a neuron with a test compound; and (b)
assaying the level of p75.sup.NTR signaling in the neuron, wherein
a decrease in p75.sup.NTR signaling relative to a neuron not
contacted with the test compound identifies the test compound as a
compound that increases neuronal growth.
[0017] The neuron can be, for example, a sympathetic neuron, a
cholinergic neuron, a sensory neuron, a cortical neuron, a motor
neuron, or a neuron derived from the dorsal root ganglia. It is
desirable that the neuron is from a mammal (e.g., a human). The
neuron can be in vitro or in vivo.
[0018] The test compound can be selected from the group consisting
of a peptide or protein, a carbohydrate, a lipid, a nucleic acid
molecule, a natural organic molecule, a synthetically derived
organic molecule, an antibody (e.g., one that binds p75.sup.NTR),
or a derivative or modification thereof.
[0019] In yet another aspect, the invention a method of treating a
patient having a neurodegenerative disease, the method including
administering to the patient a compound that increases MEK
signaling in a neuron in the patient.
[0020] In a related aspect, the invention features a method of
treating a patient having a neurodegenerative disease, the method
including administering to the patient a compound that decreases
p75.sup.NTR signaling in a neuron in the patient.
[0021] By "neuron" is meant a cell of embryonic ectodermal origin
derived from any part of the nervous system of an animal. Neurons
express well-characterized neuron-specific markers, including class
III .beta.-tubulin, MAP2, and neurofilament proteins. Neurons
include, without limitation, cortical neurons, motor neurons,
sensory neurons, cholinergic neurons, sympathetic neurons, and
neurons derived from the dorsal root ganglion. Also considered to
be a neuron is a cell expressing one or more neuron-specific
markers, regardless of the origin. For example, neurons can be
derived from cell lines (e.g., PC-12 cells, P-19 cells, and
neuroblastoma cells) and stem cells (e.g., embryonic stem cells,
mesenchymal stem cells, neural stem cells).
[0022] By "neuronal growth" is meant an increase in process network
density (e.g., axonal, dendritic, or neuritic growth, including
branching), cell migration, or target innervation. Any of these
aspect of neuronal growth may be measured using techniques known in
the art and as described herein.
[0023] By "neuronal processes" is meant the axons, dendrites, or
neurites (axons and dendrites) formed by a neuron. These are
routinely characterized (e.g., in terms of shape, number, and
targets innervated) using, for example, standard microscopic
techniques.
[0024] By "NGF" is meant nerve growth factor which, in general,
binds the TrkA receptor and promotes neuronal survival and growth
and may be purified from, e.g., mouse salivary glands or produced
recombinantly.
[0025] By "BDNF" is meant brain-derived neurotrophic factor, which,
in general, binds the p75.sup.NTR neurotrophin receptor and
inhibits neuronal growth and target innervation, or binds TrkB and
promotes neuronal growth and survival.
[0026] By "test compound" is meant a chemical, be it
naturally-occurring or artificial, that is surveyed for its ability
to modulate cell death, by employing one of the assay methods
described herein. Test compounds may include, for example,
peptides, polypeptides, antibodies (and fragments thereof),
synthesized organic molecules, naturally occurring organic
molecules, nucleic acid molecules, and components or derivatives
thereof.
[0027] By "assaying" is meant analyzing the effect of a treatment,
be it chemical or physical, administered to whole animals or cells
derived therefrom. The material being analyzed may be an animal, a
cell, a lysate or extract derived from a cell, or a molecule
derived from a cell. The analysis may be, for example, for the
purpose of detecting altered gene expression, altered RNA
stability, altered protein stability, altered protein levels,
altered protein phosphorylation, or altered protein biological
activity. The means for analyzing may include, for example,
antibody labeling, immunoprecipitation, phosphorylation assays, and
methods known to those skilled in the art for detecting nucleic
acids.
[0028] By "modulating" is meant changing, either by decrease or
increase.
[0029] By "p75.sup.NTR pathway" is meant the entire signal
transduction pathway, or part thereof, that is sufficient to
transfer a signal that inhibits neuronal growth in the presence of
NGF. Components of the p75.sup.NTR pathway that can contribute to
the positive regulation of this pathway (i.e., repress neuronal
growth) are, for example, activated MEKK, activated JNK, dominant
negative Ras (e.g., N17Ras), dominant negative Akt, c-jun, p53,
p21, and Bax.
[0030] By "Trk" is meant TrlA, TrkB, or TrkC.
[0031] By "inhibitor of MEK/MAPK pathway" is meant any compound
that inhibits MEK/MAPK-mediated neuronal growth.
[0032] By "alteration in the level of gene expression" is meant a
change in gene activity such that the amount of a product of the
gene, i.e., mRNA or polypeptide, is increased or decreased, or that
the stability of the mRNA or the polypeptide is increased or
decreased.
[0033] By "reporter gene" is meant any gene which encodes a product
whose expression is detectable and/or quantifiable by physical,
immunological, chemical, biochemical or biological assays. A
reporter gene product may, for example, have one of the following
attributes, without restriction: a specific nucleic acid/chip
hybridization pattern, fluorescence (e.g., green fluorescent
protein), enzymatic activity (e.g., lacZ/.beta.-galactosidase,
luciferase, chloramphenicol acetyltransferase), toxicity (e.g.,
ricin A), or an ability to be specifically bound by a second
molecule (e.g., biotin or a detectably labelled antibody). It is
understood that any engineered variants of reporter genes, which
are readily available to one skilled in the art, are also included,
without restriction, in the foregoing definition.
[0034] By "operably linked" is meant that a gene and a regulatory
sequence are connected in such a way as to permit expression of the
gene product under the control of the regulatory sequence.
[0035] By a "transgene" is meant a nucleic acid sequence which is
inserted by artifice into a cell and becomes a part of the genome
of that cell and its progeny. Such a transgene may be partly or
entirely heterologous to the cell, or may be in a position or under
regulatory control which is distinct from that of a naturally
occurring cell.
[0036] By "transgenic animal" is meant an animal having a transgene
as described above.
[0037] By "increase in neuronal growth" is meant an increase in the
number, length, or branching of axons, dendrites, or neurites of a
neuron compared to a control neuron. The neuron may also exhibit
other aspects of neuronal growth as described above.
[0038] By a protein or mRNA that is "preferentially found" in
dendrites is meant one that is present in a substantially greater
amount in dendrites than it is in axons. Abundance of the protein
or mRNA can be at least 5-fold greater in dendrites than it is in
axons (per unit length), or even 10-fold more abundant (or greater)
in dendrites than it is in axons. Similarly, a protein or mRNA that
is "preferentially found" in axons is one that is in a
substantially greater amount in axons than it is in dendrites,
preferably at least 5-fold and more preferably at least 10-fold
greater. A protein or mRNA that is "preferentially found" in
neurites is one that is in substantially greater amount in axons
and/or dendrites than it is in the cell soma. Again, it is
desirable that the difference be at least 5-fold, at least 10-fold,
or greater.
[0039] The methods described herein allow for the identification of
a compound that stimulates neuronal growth. Moreover, these methods
are readily adapted for high throughput drug screening.
[0040] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIGS. 1A-1C are schematic illustrations that show that BDNF
signaling through p75.sup.NTR decreases the growth of sympathetic
neurons in vitro without affecting their survival. FIG. 1A shows
results of colorimetric MTT assays to measure mitochondrial
function and cell survival. FIGS. 1B and 1C show quantitative
analysis of neurite process density in sympathetic neuron cultures
grown in the presence of NGF or NGF plus BDNF.
[0042] FIGS. 2A-2D are photographs that show that exogenous BDNF
inhibits and anti-BDNF and anti-p75.sup.NTR each enhance
NGF-promoted growth of sympathetic neurons in vitro.
[0043] FIG. 3 is a photograph of an immunoblot that shows that a
function-blocking BDNF antibody neutralizes the ability of
exogenous BDNF to activate TrkB.
[0044] FIGS. 4A-4F are schematic illustrations that show that
function-blocking antibodies directed against BDNF or p75.sup.NTR
enhance growth of sympathetic neurons without affecting their
survival.
[0045] FIG. 4A shows results of colorimetric MTT assays to measure
mitochondrial function and cell survival.
[0046] FIGS. 4B-4F depict quantitative analysis of neuritic process
density in sympathetic neuron cultures grown in the presence of
NGF, NGF plus anti-BDNF (FIGS. 4B and 4C), NGF plus
anti-p75.sup.NTR (FIGS. 4D and 4E), or increased NGF (FIG. 4F).
[0047] FIGS. 5A-5D are schematic illustrations that depict an
analysis of neurite outgrowth in response to NGF or NGF plus BDNF
in p75.sup.NTR-/- versus p75.sup.NTR+/+ sympathetic neurons.
[0048] FIGS. 5A and 5B show results of colorimetric MTT assays to
measure mitochondrial function and survival of murine sympathetic
neurons in response to NGF or NGF plus BDNF.
[0049] FIGS. 5C-5D show quantitative analysis of neuritic process
density in p75.sup.NTR-/- versus wild-type murine sympathetic
neurons in response to NGF or NGF plus BDNF.
[0050] FIGS. 6A-6D are photographs that show that p75.sup.-/-
sympathetic neurons show enhanced neuritogenesis in response to
NGF, but do not respond to exogenous BDNF. The phase contrast
micrographs are of cultured Coomassie Blue-stained wild-type (FIGS.
6A-6B) and p75.sup.NTR.sup.-/- murine sympathetic neurons (FIGS.
6C-6D) maintained in 50 ng/ml NGF for two days, and then switched
to 7.5 ng/ml NGF (FIGS. 6A and 6C), or 7.5 ng/ml NGF plus 100 ng/ml
BDNF (FIGS. 6B and 6D).
[0051] FIGS. 7A-7E are photographs of immunoblots that show BDNF
and p75.sup.NTR levels in transgenic mice and that BDNF and
p75.sup.NTR are present in the pineal gland during the period of
sympathetic target innervation.
[0052] FIG. 7A represents immunoblot analysis for BDNF in the adult
rat pineal gland.
[0053] FIG. 7B represents an immunoblot analysis for BDNF in the
pineal gland of BDNF.sup.-/-, BDNF.sup.+/-, and BDNF.sup.+/+ litter
mates at P13 to P15.
[0054] FIG. 7C represents an immunoblot analysis for p75.sup.NTR in
the developing postnatal rat pineal gland.
[0055] FIGS. 7D and 7E show that levels of tyrosine hydroxylase, a
specific marker for sympathetic fibers, are increased in the pineal
glands of BDNF.sup.+/- and BDNF.sup.-/- mice.
[0056] FIG. 7D shows an immunoblot analysis of tyrosine
hydroxylase, a specific marker for sympathetic fibers, in equal
amounts of protein from the pineal glands of BDNF.sup.+/+,
BDNF.sup.+/-, and BDNF.sup.-/- litter mates at P13 to P15.
[0057] FIG. 7E shows an immunoblot analysis of p75.sup.NTR in equal
amounts of protein from the pineal glands of BDNF.sup.+/+,
BDNF.sup.+/-, and BDNF.sup.-/- litter mates at P13 to P15.
[0058] FIGS. 8A-8J are photographs that show that the pineal gland
is hyperinnervated with sympathetic fibers in BDNF.sup.-/- mice at
P13. The photographs are of immunohistochemical analysis for
tyrosine hydroxylase, a specific marker for sympathetic fibers, in
sections of the pineal gland from a control litter mate (FIGS. 8A,
8C-8E) relative to a BDNF.sup.-/- litter mate (FIGS. 8B,
8F-8H).
[0059] FIG. 9A is a schematic illustration of MEK isoforms. DN MEK
is a dominant-negative MEK isoform, while G1C (ACT MEK) is
constitutively active. Also shown is a schematic illustration of an
adenoviral vector carrying MEK and GFP.
[0060] FIG. 9B is a series of photographs of immunoblots showing
that adenovirally expressed DN MEK decreased MAPK phosphorylation
in both PC 12 cells and SCG neurons.
[0061] FIG. 9C is a photograph of immunocytochemical detection of
adenovirally infected cells expressing DN MEK.
[0062] FIG. 9D is a schematic illustration showing that none of the
MEK isoforms increased SCG neuronal survival in the absence of
NGF.
[0063] FIG. 10A is a schematic illustration of a chamber used in
neuronal growth assays.
[0064] FIG. 10B is a series of photographs showing tubulin staining
of hepatocyte growth factor (HGF)-induced neurites from DN
MEK-expressing neurons (right panel) or control neurons (left
panel).
[0065] FIG. 10C is a quantification of the neuritic growth
exemplified in FIG. 10B.
[0066] FIG. 11 is a photograph showing the expression pattern of
reporter gene operably linked to the T.alpha.1 .alpha.-tubulin
promoter.
[0067] FIG. 12 is a schematic illustration showing that in vivo
expression of DN MEK decreases P-MAPK levels in the
hippocampus.
[0068] FIGS. 13A and 13B are schematic illustrations showing that
expression of DN MEK decreases innervation of the pineal gland by
sympathetic neurons by approximately 75% (FIG. 13A) without a
reduction in the number of SCG neurons (FIG. 13B).
[0069] FIGS. 14A and 14B are a series of photographs and a
schematic, respectively, showing results with DN MEK.
[0070] FIG. 14A is a photograph showing the medial branch of the
dorsal cutaneous nerve having selective loss of the unmyelinated
C-fibers in mice expressing DN MEK (left), relative to the control
(right).
[0071] FIG. 14B is a schematic illustration showing that expression
of DN MEK results in a loss of C-fibers in the dorsal cutaneous
nerve.
[0072] FIG. 15 is a schematic illustration showing that MEK
inhibitor U0126 blocks NGF/TrkA-mediated neurite outgrowth in
primary sympathetic neurons.
[0073] FIG. 16 is a schematic illustration showing that U0126
blocks BDNF/TrkB-mediated neurite outgrowth in primary sympathetic
neurons that were made to exogenously express TrkB by infection
with a recombinant adenovirus.
[0074] FIGS. 17A and 17B are schematic illustrations showing that a
mutant TrkB receptor, exhibiting reduced activation of MEK, is
severely deficient in promoting neurite outgrowth.
[0075] FIG. 18 is a schematic illustration showing that inhibition
of MEK activity by overexpression of DN MEK attenuated
TrkB-mediated neurite outgrowth in primary sympathetic neurons made
to exogenously express TrkB by infection with a recombinant
adenovirus.
[0076] FIGS. 19A-19C are photographs showing HMW-MAP2 localization
following treatment for three days with 10 ng/ml NGF (FIG. 19A), 50
mM KCl (FIG. 19B), or 10 ng/ml NGF plus 50 mM KCl (FIG. 19C). Scale
bar=100 .mu.M.
[0077] FIGS. 20A and 20B are high magnification photogrpahs of MAP2
staining of SCG neurons in the presence of NGF (FIG. 20A) and
NGF+KCl (FIG. 20B). Scale bar=50 .mu.M.
[0078] FIG. 21 is a schematic illustration showing image analysis
of cultures shown in FIGS. 19A-19C. Lengths of individuals
dendrites were counted and histogram analysis was performed using
bin sizes of 2 .mu.M. The histogram analyses were then normalized
so that the population data could be compared between different
treatments.
[0079] FIG. 22 is a schematic illustration showing immuno-dot blot
analysis of MAP2 association and tubulin dynamics in SCG neurons.
The relative amounts of MAP2 present per .mu.g of assembled tubulin
(relative association of MAP2 with microtubules) were determined by
the ratio of AP20 signal (dendrite specificMAP2) to DM1A signal
(polymerized tubulin) following three days of treatment with 10
ng/ml NGF, 50 mM KCl, or 10 ng/ml NGF plus 50 mM KCl.
[0080] FIGS. 23A-23D are a series of photographs showing MAP2
immunofluorescence in SCG neurons treated for three days with 50 mM
KCl (FIG. 23A), followed by two days treatment with 10 ng/ml NGF
alone (FIG. 23B) or 10 ng/ml NGF plus 50 mM KCl (FIG. 23C) followed
by two days treatment with 10 ng/ml NGF alone (FIG. 23D). Scale
bar=100 .mu.M.
[0081] FIGS. 24A-24J are a series of photographs showing MAP2
immunofluorescence in SCG neurons treated for three days with 10
ng/ml NGF (FIGS. 24A-24E) or 10 ng/ml NGF plus 50 mM KCl (FIGS.
24F-24J). Cultures were simultaneously exposed to 10 .mu.M CaMKII
inhibitor KN-62 (FIG. 24B, 24G), 50 .mu.M MEK inhibitor PD98059
(FIGS. 24C, 24H), 100 nM PKA inhibitor H-89 (FIGS. 24D, 24I) or 10
.mu.M adenylate cyclase inhibitor Forskolin (FIGS. 24E, 24J).
Control cultures are shown in FIGS. 24A and 24F. Scale bar 100
.mu.M.
[0082] FIG. 25 is a schematic illustration showing image analysis
of MAP2 localization in the SCG cultures shown in FIGS. 24A-24J.
Note that inhibitation of CaMKII or MEK activities resulted in a
suppression of dendritic maturation in response to KCl.
[0083] FIG. 26 is a schematic illustration showing immuno-dot blot
analysis of MAP2 association and microtubule dynamics in the SCG
cultures shown in FIGS. 24A-24J. The data represent the effect of
kinase inhibitors on the relative amounts of MAP2 associated per
.mu.g of assembled tubulin.
[0084] FIG. 27 is a schematic illustration showing biochemical
analysis of the effects of NGF, KCl, and kinase inhibitors on MAP2
levels. Note both KN-62 and PD98059 reduce MAP2 levels.
DETAILED DESCRIPTION
[0085] We have discovered that Trk and p75.sup.NTR have
functionally antagonistic actions on neuron growth and target
innervation, with NGF acting via TrkA to promote growth and BDNF
via p75.sup.NTR to inhibit growth. Specifically, in rat sympathetic
neurons (which do not express TrkB), BDNF signals through
p75.sup.NTR to inhibit neuronal growth. Similarly,
function-blocking BDNF antibodies can enhance neuronal growth of
these cells. Both exogenous and autocrine BDNF mediate this effect
via p75.sup.NTR since (i) BDNF does not inhibit growth of neurons
lacking p75.sup.NTR; (ii) function-blocking p75.sup.NTR antibodies
enhance NGF-mediated growth, and; (iii) p75.sup.NTR-/- sympathetic
neurons grow better in response to NGF than do their wild-type
counterparts. Thus, BDNF, made by sympathetic neurons and/or their
target organs, acts via p75.sup.NTR to antagonize NGF-mediated
growth and target innervation, indicating that sympathetic target
innervation is determined by the balance of positively- and
negatively-acting neurotrophins present in developing, and
potentially, mature targets.
[0086] We have also discovered that two kinases, MAP kinase (MAPK)
and MAP kinase kinase (MEK), transduce Trk-mediated neuronal growth
signals, but are not required for NGF-mediated cell survival. Thus,
any compound that blocks p75.sup.NTR signaling or promotes MEK/MAPK
signaling is expected to promote neuronal growth of sympathetic
neurons. Accordingly, we have developed assays in which neuronal
growth is blocked either by the activation of the p75.sup.NTR
pathway, or by disruption of MEK/MAPK signaling. These assays can
be employed to identify compounds that overcome the signals
inhibiting neuronal growth and thus stimulate neuronal growth and
target innervation. The identified compounds are likely to be
genetically downstream of both p75 and MEK/MAPK and, thus, are
expected to be selective for promoting neuronal growth. Compounds
identified using the screens described herein are useful for
treating neurodegenerative disease (e.g., Alzheimer's disease,
Huntington's disease, Parkinson's disease, and amyotrophic lateral
sclerosis) or disease resulting from trauma, such as ischemic
stroke or axotomy.
[0087] We believe this functional antagonism between Trk-mediated
MEK signaling and p75.sup.NTR signaling may be generalized to
neurons other than sympathetic neurons. Evidence for this is found
in p75.sup.NTR-/- mice, in which the number of basal forebrain
cholinergic neurons increases and the hippocampus is
hyperinnervated, a phenotype reminiscent of what is seen for
sympathetic neurons in p75.sup.NTR-/- and BDNF.sup.-/- mice.
Moreover, this same functional antagonism may also occur in adult
sensory neurons, as well as in neurons of the dorsal root
ganglia.
[0088] Without wishing to be bound to a particular theory, we
believe that p75.sup.NTR inhibits Trk-mediated neuronal growth
through p75.sup.NTR-mediated generation of intracellular ceramide.
Thus, activation of p75.sup.NTR could well attenuate neurite growth
via increased ceramide.
[0089] We have also discovered that inhibition of MEK signaling
(e.g., through the expression of a dominant-negative MEK (DN MEK)
or the administration of U0126, a specific inhibitor of MEK)
interferes with Trk-mediated cell growth without perturbing cell
survival. For example, transgenic animals expressing DN MEK exhibit
a decrease of neuronal growth in the pineal gland. Thus, activation
of p75.sup.NTR and suppression of MEK signaling produce a similar
phenotype.
[0090] The present discoveries can be exploited in useful
high-throughput screening assays for compounds that stimulate
neuronal growth and target innervation. Development of assays for
compounds which modulate neuronal growth and target innervation
with high specificity requires, first, identification of key,
specific steps in the neuronal growth pathway and, second,
development of assays to measure changes at these steps. To
determine the mechanism whereby the neurotrophins stimulate the
survival and growth of normal and injured neurons, we have asked
whether specific components of the p75.sup.NTR and MEK/MAPK
pathways play a role in modulating neuronal growth, focusing our
studies on cultured rat sympathetic cervical ganglion (SCG) neurons
in order to determine the exact nature of this regulation. In
addition, we have taken advantage of transgenic mice lacking either
BDNF or p75.sup.NTR or, alternatively, expressing a dominant
negative MEK to demonstrate the role these factors play in vivo.
The examples below show that alterations in the signaling of each
of these pathways alters neuronal growth. These findings serve as a
basis for the development of assays for the identification of
compounds that modulate one or both signaling pathways and, thus,
modulate neuronal growth.
[0091] Primary Screens for Compounds that Modulate the
BDNF/p75.sup.NTR or MEK/MAPK Pathway and Neuronal Growth
[0092] We have discovered that both the p75.sup.NTR pathway and the
MEK/MAPK pathway modulate neuronal growth. These findings allow us
to provide assays for drugs which affect neuronal growth by
modulation of these two signaling pathways. Such assays may measure
various aspects of signaling components that include changes in:
(a) phosphorylation status; (b) kinase activity; (c) neuronal
growth; and (d) mRNA or polypeptide levels of components of either
the p75.sup.NTR or MEK/MAPK pathway. These measurements, which can
be made in vitro or in vivo, form the basis of assays which
identify compounds that modulate neuronal growth. Such identified
compounds may have therapeutic value, for example, in the treatment
of neurodegenerative disease and neurological trauma.
[0093] Secondary Screens for Compounds that Modulate Neuronal
Growth.
[0094] After test compounds that appear to have neuronal
growth-modulating activity are identified, it may be necessary or
desirable to subject these compounds to further testing. The
invention provides such secondary confirmatory assays. For example,
a compound that appears to increase neuronal growth in early
testing is subject to additional assays to confirm that the levels
of other cell markers of neuronal growth are reproducibly
influenced by the compound. At late stages, testing is performed in
vivo to confirm that the compounds initially identified to affect
neuronal growth in cultured neurons has the predicted effect on in
vivo neurons. In the first round of in vivo testing, neuronal
growth is conducted in animals using well-known methods and then
the compound is administered by one of the means described in the
"Therapy" section immediately below. Results may be compared to
transgenic mice lacking BDNF and/or p75.sup.NTR, or those in which
MEK/MAPK signaling has been inhibited. Neurons or neural tissue are
isolated within hours to days following the insult, and are
subjected to assays as described in the examples below. Such assays
are well known to those skilled in the art. Examples of such assays
include, but are not limited to: immunolabeling, measuring
neurotransmitter levels, ceramide levels, or phosphorylation
levels, or monitoring a change in the shape, number, or otherwise
growth-related quantifiable characteristic exhibited by the
neuron.
[0095] In addition to SCG neurons or other sympathetic neurons,
other types of primary neurons, isolated by standard techniques,
such as cortical neurons, hippocampal neurons, and motor neurons,
may also be used (see, e.g., Brewer et al., Nature 363:265-266,
1993; Henderson et al., Nature 363:266-267, 1993).
[0096] Cellular levels of other biochemical markers may be employed
as an indication that neuronal growth is modulated by a test
compound. Measurement of a polypeptide, mRNA, PCR product, and
reporter gene activity of a reporter operatively linked to a
protein promoter involved in either the p75.sup.NTR pathway or the
MEK/MAPK pathway are also part of the invention.
[0097] The assays described herein can also be used to test for
compounds that inhibit neuronal growth and increase cell death and
hence may have therapeutic value in the treatment of
neuroproliferative disease.
[0098] Test Compounds
[0099] In general, novel drugs that promote neuronal growth are
identified from large libraries of both natural product or
synthetic (or semi-synthetic) extracts or chemical libraries
according to methods known in the art. Those skilled in the field
of drug discovery and development will understand that the precise
source of test extracts or compounds is not critical to the
screening procedure(s) of the invention. Accordingly, virtually any
number of chemical extracts or compounds can be screened using the
exemplary methods described herein. Examples of such extracts or
compounds include, but are not limited to, plant-, fungal-,
prokaryotic- or animal-based extracts, fermentation broths, and
synthetic compounds, as well as modification of existing compounds.
Numerous methods are also available for generating random or
directed synthesis (e.g., semi-synthesis or total synthesis) of any
number of chemical compounds, including, but not limited to,
saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
Synthetic compound libraries are commercially available, for
example, from Brandon Associates (Merrimack, N.H.) and Aldrich
Chemical (Milwaukee, Wis.). Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant, and animal
extracts are commercially available from a number of sources,
including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch
Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A.
(Cambridge, Mass.). In addition, natural and synthetically produced
libraries are produced, if desired, according to methods known in
the art, e.g., by standard extraction and fractionation methods.
Furthermore, antibodies (or fragments thereof) may also be
generated using standard techniques known in the art and screened
for their efficacy in promoting neuronal growth using the
techniques described herein.
[0100] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication
(e.g., taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their therapeutic activities for neurodegenerative disorders should
be employed whenever possible.
[0101] When a crude extract is found to increase neuronal growth,
further fractionation of the positive lead extract is necessary to
isolate chemical constituents responsible for the observed effect.
Thus, the goal of the extraction, fractionation, and purification
process is the careful characterization and identification of a
chemical entity within the crude extract having neuronal growth
promoting activities. The same assays described herein for the
detection of activities in mixtures of compounds can be used to
purify the active component and to test derivatives thereof.
Methods of fractionation and purification of such heterogenous
extracts are known in the art. If desired, compounds shown to be
useful agents for treatment are chemically modified according to
methods known in the art. Compounds identified as being of
therapeutic value may be subsequently analyzed using a mammalian
neuronal growth model.
[0102] Below are examples of assays that are readily adapted to
high-throughput systems useful for evaluating the efficacy of a
molecule or compound useful in promoting neuronal growth.
[0103] Therapy
[0104] Compounds identified using any of the methods disclosed
herein may be administered to patients or experimental animals with
a pharmaceutically-acceptable diluent, carrier, or excipient, in
unit dosage form. Conventional pharmaceutical practice may be
employed to provide suitable formulations or compositions to
administer such compositions to patients or experimental animals.
Although intravenous administration is preferred, any appropriate
route of administration may be employed, for example, parenteral,
subcutaneous, intramuscular, intracranial, intraorbital,
ophthalmic, intraventricular, intracapsular, intraspinal,
intracistemal, intraperitoneal, intranasal, aerosol, or oral
administration. Therapeutic formulations may be in the form of
liquid solutions or suspensions; for oral administration,
formulations may be in the form of tablets or capsules; and for
intranasal formulations, in the form of powders, nasal drops, or
aerosols.
[0105] Methods well known in the art for making formulations are
found, for example, in Remington: The Science and Practice of
Pharmacy, (19th ed.) ed. Gennaro A R., 1995, Mack Publishing
Company, Easton, Pa. Formulations for parenteral administration
may, for example, contain excipients, sterile water, or saline,
polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, or hydrogenated napthalenes. Biocompatible, biodegradable
lactide polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for antagonists or agonists of the invention
include ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Formulations for
inhalation may contain excipients, for example, lactose, or may be
aqueous solutions containing, for example, polyoxyethylene-9-lauryl
ether, glycocholate and deoxycholate, or may be oily solutions for
administration in the form of nasal drops, or as a gel. In
addition, specific techniques may be employed for delivering
molecules across the bloodbrain barrier (BBB) of the central
nervous system (CNS). For example, shunts for direct infusion into
the brain may be employed as well as osmotic or pharmacologic
disruption of the BBB (see, e.g., Kroll et al., Neurosurgery
42:1083-1100, 1998).
[0106] The following examples are provided to illustrate the
invention and should not be construed as limiting.
EXAMPLE 1
General Methods
[0107] Neuronal Cell Culture
[0108] Mass cultures of pure sympathetic neurons from the superior
cervical ganglion (SCG) of postnatal day 1 Sprague Dawley rats
(Charles River Breeding Laboratories, St. Constant, Quebec, Canada)
were prepared as previously described (Ma et al., J. Cell Biol.
117:135-141, 1992). Neurons were plated at low density
(approximately one ganglion/well) in Nunclon.TM. 4-well culture
dishes (Gibco BRL, Burlington, Ontario, Canada), coated with either
rat tail collagen or poly-D-lysine and laminin (both from
Collaborative Biomedical Products, Bedford, Mass.). Culture medium
was UltraCulture (BioWhittaker, Walkersville, Md.), supplemented
with 3% rat serum (Harlan Bioproducts, Madison, Wis.), 2 mM
glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin (all from
BioWhittaker), and for the first two days, 7 .mu.M cytosine
arabinoside (Sigma-Aldrich Canada Ltd., Oakville, Ontario,
Canada).
[0109] CD-1 mouse sympathetic neurons were cultured by a
modification of the method used to prepare rat neurons. Mouse
cultures were essentially prepared the same way, but were
dissociated in UltraCulture medium rather than Hanks' balanced
saline solution. 3% fetal bovine serum (Gibco) was used instead of
rat serum, and 3.5 .mu.M cytosine arabinoside was added to the
culture medium on day one post-plating.
[0110] NGF used in these experiments was purified from mouse
salivary glands and as supplied by Cedarlane Laboratories Ltd.
(Hornby, Ontario, Canada). The sources of recombinant human BDNF
were PeproTech Inc. (Rocky Hill, N.J.), for the neuritogenesis
assays, and Promega Corporation, (Madison, Wis.), for the rhBDNF
neutralization experiments. The p75.sup.NTR function-blocking
antibody REX is directed against the extracellular domain of
p75.sup.NTR, and was used as an antiserum at a dilution of 1:100
(Weskamp and Reichardt, Neuron 6:649-663, 1991). Rabbit serum
(Gibco) of the same concentration was used as the negative control
for REX. Anti-Human BDNF pAb (Promega), was used at 10 .mu.g/ml. As
a negative control for anti-BDNF, nonimmune chicken IgY (Promega)
was used at up to 40 .mu.g/ml in BDNF neutralization experiments,
and 10 .mu.g/ml in neuritogenesis experiments.
[0111] BDNF Neutralization
[0112] To test the capacity of anti-BDNF to neutralize BDNF,
TrkB-expressing NIH-3T3 cells were cultured in Dulbecco's modified
Eagle medium. Briefly, cells were washed twice and incubated for
one hour at 37.degree. C. in buffer, followed by a 30 minute wash
at 37.degree. C. in a phosphate-free buffer. Treatment consisted of
incubating cells for five minutes with either 50 ng/ml BDNF
(Promega) or with BDNF preabsorbed for four hours at 4.degree. C.
with increasing concentrations of the BDNF antibody (5-40
.mu.g/ml). In addition, TrkB-3T3 cells were treated with medium
only, or medium plus 40 .mu.g/ml nonimmune chicken IgY. Following
these treatments, cells were lysed, immunoprecipitated with
anti-pan Trk (Hempstead et al., Neuron 9:883-896, 1992), and the
immunoprecipitates analyzed for TrkB activation by Western blot
analysis with phosphotyrosine antibody 4G10 (Upstate Biotechnology
Inc., Lake Placid, N.Y.).
[0113] Neuronal Cell Survival Assays
[0114] NGF-dependent neurons were selected by culturing sympathetic
neurons for five days in the presence of 50 ng/ml NGF. Neurons were
washed three times for one hour each in neurotrophin-free media,
and were then fed with media containing 10 ng/ml NGF alone, or
various concentrations of NGF plus either 100 ng/ml BDNF, 10
.mu.g/ml .alpha.BDNF, or a 1:100 dilution of antiserum containing
the p75.sup.NTR antibody, REX (Weskamp and Reichardt, Neuron 6:
649-663, 1991). Each condition was repeated in triplicate, and
analysis of survival was performed 48 hours later by using
nonradioactive cell proliferation (MTT) assays (Celltitre 96;
Promega; Belliveau et al., J. Cell Biol. 136:375-388, 1997).
Specifically, 50 .mu.l of the MTT reagent was added to 500 .mu.l
media in each well, and incubated for two hours at 37.degree. C.
After aspiration of the MTT containing media, 100 .mu.l of a 0.065N
HCl/isopropanol mixture was added to each well to lyse the cells,
and colorimetric analysis was performed using an ELISA reader. For
rat sympathetic neurons, 10 ng/ml NGF represents 100% survival,
therefore all other values were considered to be relative to 10
ng/ml NGF. For mouse neurons, 7.5 ng/ml NGF represents 100%
survival, and was thus considered to be the 100% survival threshold
for neuritogenesis experiments.
[0115] Analysis of Transgenic Animals
[0116] Mice heterozygous for a targeted mutation in the BDNF gene
or homozygous for a targeted mutation in the p75.sup.NTR gene were
obtained from Jackson Laboratories (Bar Harbor, Me.). The
BDNF.sup.-/- mice were maintained in a C129/BalbC background. The
p75.sup.NTR-/- mice were originally generated in a C129 background,
and were backcrossed into a C129 background before purchase from
Jackson Laboratories and then maintained as homozygotes. Progeny
from BDNF heterozygote crosses were screened for the mutant
allele(s) using PCR.
[0117] Quantification of Neuronal Growth
[0118] Regulation of neuronal growth was analyzed by an in vitro
neuritogenesis assay. Postnatal day one rat sympathetic neurons
were cultured in 50 ng/ml NGF for two to three days in order to
upregulate p75.sup.NTR, whose increased expression in response to
NGF occurs independently of neuronal survival. Following a one hour
wash in neurotrophin-free medium, cultures were maintained for an
additional two days in 10 ng/ml NGF plus or minus 100 ng/ml BDNF.
Fields in sister cultures containing a similar number of neuronal
cell bodies were then photographed, and neuritic process density
was determined as described below. Since murine sympathetic neurons
are more sensitive to NGF, requiring less to maintain 100% survival
in vitro, experiments using mouse neurons were performed with a
background of 7.5 ng/ml NGF in the medium instead of 10 ng/ml NGF,
and were stained with Coomassie Blue for five minutes before being
photographed.
[0119] Quantitative analysis was performed by using common
statistics applied to random sets of lines and determining the
number of intersection points per unit area. The network of
neuritic processes, when viewed microscopically, appears as a
random set of lines in a plane. Low density fields were
photographed, and the number of visible intersections and
bifurcations of cell processes per unit area was considered to be a
quantitative measure of process density. Since the number of
neurites is a direct function of the number of neurons, however,
the number of intersections and bifurcations counted per unit area
was normalized to the number of cell bodies in that same area of
interest. For each experimental condition, 6-8 sampling windows (10
mm.sup.2) were photographed, and the Student's t test was used to
determine the statistical significance of density differences
between experimental groups. Results are expressed as the mean
density plus or minus the standard error of the mean.
[0120] Another assay to assess neuronal growth is based upon the
measurement of tubulin, the major protein component of neurites and
axons. In one assay, neurons are lysed and quantitative dot blots
are performed. Using this approach, we have demonstrated that there
is a correlation between the amount of tubulin and the amount of
growth, as measured using morphological criteria. ELISA, using the
same tubulin antibody, may also be used.
[0121] A further assay to measure neuronal growth is performed as
follows. Axons of cultured sympathetic neurons are immunostained
with anti-tubulin antibody, and the percentage of surface area that
is covered by tubulin-immunoreactivity is then measured. In a
typical protocol, sympathetic neurons are grown for two days in NGF
at similar densities on collagen-coated 48- or 96-well plates. They
are then switched into 10 ng/ml NGF or 10 ng/ml NGF plus 100 ng/ml
BDNF. Two days later, they are immunostained for tubulin and
counterstained with Hoechst. The percentage area covered by tubulin
and the number of Hoechst-positive nuclei are enumerated using an
image analysis machine. Such an analysis provides
statistically-significant data collection on the amount of neurite
growth per neuron. For high-throughput screens, a plate reader may
be employed to "read" the fluorescence from the two
fluorochromes.
[0122] For illustrative purposes, two specific examples are
provided below. These are not meant to be limiting.
[0123] High-Throughput Screening of Neuronal Growth
[0124] Assays for neuronal growth (e.g., neuritic growth, axonal
growth, or dendritic growth) may, for example, be performed as
follows. Ninety-six-well plates are coated for at least one hour
with rat tail collagen (prepared in a dilution of 1% glacial acetic
acid). After one hour, excess collagen at aspirated and allow to
dry completely. Neurons (e.g., SCG neurons, or any other primary
neuron or neuron derived from a cell line or stem cell) are placed
into culture. At the desired endpoint of the growth experiment,
neurons are washed briefly with PBS and then fixed for 10 minutes
with 100 .mu.l 4% formaldehyde (freshly prepared, BDH). Excess
fixative is removed from cells by 3.times.5 min washed with 200
.mu.l PBS. PBS is aspirated and replaced with 100 .mu.l of staining
solution (e.g., 10% acetic acid, 40% methanol, 0.25% (w/v)
Coomassie Brilliant Blue R-250; "Coomassie Blue") for 15 minutes.
If Coomassie Blue is used, unbound stain can be removed by, for
example, 3.times.10 min washed with 200 .mu.l destaining solution
(10% acetic acid, 40% methanol). Remaining wash solution is
aspirated and 100 .mu.l of solubilization solution is added (10%
HCl in isopropanol). The plates can be gently agitated for five
minutes to ensure complete solubilization. The concentration of
bound dye (which is proportional to neuronal growth) is then
measured on a absorbance spectrophotometer at the appropriate
wavelength (e.g., 595 nm for Coomassie Blue).
[0125] Measurement of Dendritic Growth
[0126] Dendritic growth can be measured as follows. Ninty-six-well
plates are coated for at least 24 hours with rat tail collagen
(prepared in a dilution of 1% glacial acetic acid). After one hour,
excess collagen is aspirated and allow to dry completely. SCG
neurons are dissected, triturated and plated. Cells may also be
plated onto 8-well chamber slides. Cells are fed after about three
days and, after about five days, are switched to either 50 mM KCl
or 10 ng/ml NGF+50 mM KCl for about three additional days. Neurons
are washed briefly with PBS and then fixed for 10 minutes with 4%
formaldehyde in PBS (freshly prepared). Excess fixative is removed
by 3.times.5 min washes with PBS. Cells are stained for one hour at
room temperature with the following antibodies with 3.times.5 min
PBS washes after each incubation: primary antibody--mouse
monoclonal IgG1 anti high molecular weight MAP2 (clone AP20, from
Sigma), secondary antibody--donkey anti mouse CY3, minimum cross to
mouse and rabbit (Jackson Immunochemicals). Cell are mounted and at
least three fields of view are captured using an upright
fluorescence microscope using 530 excitation/580 emission filters.
For each individual neuron in a given field (minimum 250 neurons
total), the distance of MAP2 positive staining from the perimeter
of the cell body is measured. These measurements are performed and
tabulated using NIH image. The tabulated data are then expressed as
a population histogram, binning all the measured lengths into 2
.mu.m bins. This analysis gives an overall measure of the extent of
dendritic differentiation in the population.
[0127] The growth assay utilizes the detection of protein or mRNA
as a measure of the amount of neuronal growth. Thus any reagent
that is suitable for the detection of protein will also be suitable
for use in the present methods. Reagents suitable for a
high-throughput growth assay include, for example, Bradford/Lowry
reagent, BCA reagent, Commassie Brilliant Blue G-250 and R-250,
colloidal Coomassie Blue, and sulforhodamine 101 (which is
fluorescent). Other dyes are described in chapter 9 of the
Molecular Probes Handbook (http://www.molecularprobes.co- m).
Antigens suitable for labeling neurites include, for example, alpha
or beta tubulin, GAP43, neurofilament (light, medium and heavy
forms), MAP1B, MAP4, and juvenile MAP2 (e.g., MAP2C, MAP2D).
Antigens that are preferentially found in dendrites include, for
example, adult MAP2 (MAP2A, MAP2B), adult MAP2 mRNA, dendrin,
dendrin mRNA, polyribosomal stain (Nissel stain), CaMKII mRNA,
microtubules of mixed polarity (visualized by tannic acid fixation
under electron microscopy), mGluR1 a receptor, mGluR2 receptor, and
the 5-HT1a receptor. Note that several mRNAs (such as those listed
above) are specifically located in dendrites, and these thus serve
as markers of dendritic growth. Antigens that are preferentially
found in axons include, for example, phosphorlated MAP1B, unipolar
microtubules, 5-HT1b receptor, and phosphorylated neurofilament
(heavy form only).
[0128] Those in the art will recognize that any neuronal culture
method is compatile with the screening methods described herein,
including dissociated neurons, organotypic neuronal cultures, and
reaggregate neuronal cultures. As described above, any neuron or
neuronal cell line that can be cultured and that forms neurites
(e.g., axons, dendrites, or both) is suitable for use in the
methods described herein.
[0129] Immunohistochemistry
[0130] For analysis of sympathetic innervation density in pineal
glands of transgenic mice and control litter mates, animals were
anaesthetized with sodium pentobarbital (35 mg/kg) or isofluorane,
and killed by decapitation. Pineal glands were removed immediately
and processed for immunohistochemistry as follows: tissue was
immersion-fixed overnight (at 4.degree. C.) in 4% formaldehyde in
phosphate buffer (PB, pH 7.4), and subsequently cryoprotected in
graded sucrose solutions. Twelve micron sections were cut on a
cryostat and thaw-mounted onto chromalum-subbed slides. Sections
were post-fixed in 4% formaldehyde in PB for 10 minutes at room
temperature, and then washed for 10 minute in phosphate buffered
saline (PBS, pH 7.4). Following non-specific blocking with 4% goat
serum and 4% rat serum (both from Jackson ImmunoResearch
Laboratories, West Grove, Pa.) plus 0.2% Triton X-100 in PBS (pH
7.4), pineal sections were incubated overnight at 4.degree. C. with
a commercially available polyclonal antibody directed against
tyrosine hydroxylase (1:400, Chemicon International, Temecula,
Calif.) in blocking solution. Slides were then washed three times
(10 minutes each) in PBS, and incubated for two hours in blocking
solution containing a CY3-conjugated secondary antibody (goat
anti-rabbit IgG, 1:2000, Jackson ImmunoResearch). Following
3.times.10 minute washes in PBS, slides were coverslipped using
Sigma Mounting Medium (Sigma Diagnostics, St. Louis, Mo.), and
innervation density viewed by epifluorescence microscopy. For
analysis of p75.sup.NTR levels in sympathetic terminals in the
pineal gland, pineal glands from P6 or adult Sprague Dawley rats
were processed and visualized as above, but the primary antibody
used was MC192 (Chandler et al., J. Biol. Chem. 259:6882-6889,
1984), which is directed against the extracellular domain of
p75.sup.NTR. The secondary antibody was a CY3-conjugated goat
anti-mouse IgG used at a concentration of 1:2000 (Jackson
Immunoresearch).
[0131] Immunoblot Analysis
[0132] For biochemistry, pineal glands or cortices were homogenized
in Tris buffered saline (TBS) containing 137 mM NaCl, 20 mM Tris
(pH 8.0), 1% v/v NP-40, 0.1% SDS, 10% glycerol and the protease
inhibitors phenylmethyl sulfonyl fluoride (PMSF, 1 mM), aprotinin
(10 .mu.g/ml), leupeptin (0.2 .mu.g/ml), and sodium vanadate (1.5
mM). The tissue was rocked for 10 minutes at 4.degree. C., and
following a 10 minute centrifugation at 4.degree. C., the
supernatant was collected and lysates normalized for protein
concentration using a BCA Protein Assay kit (Pierce, Rockford,
Ill.). p75.sup.NTR protein was immunoprecipitated with of
anti-recombinant human p75.sup.NTR (Promega) per 500 .mu.l lysis
buffer. The immunoprecipitates were collected with protein
A-Sepharose.TM. beads (Pharmacia Biotechnology Inc., Piscataway,
N.J.) for 1.5 hours at 4.degree. C., followed by centrifugation.
For immunoblot analysis, equal amounts of protein were boiled in
sample buffer for five minutes, and separated by 7.5% or 15%
SDS-PAGE (7.5% for tyrosine hydroxylase and p75.sup.NTR, and 15%
for BDNF). After electrophoresis, proteins were transferred onto
0.2 .mu.m nitrocellulose membranes for 1.5 hours at 0.6 amps, and
washed three times (10 minutes each) with PBS. Following a 1.5 hour
block in blotto (3% nonfat milk in PBST) at room temperature,
membranes were incubated overnight at 4.degree. C. in blocking
solution containing either anti-BDNF (1:3000; Santa Cruz
Biotechnology Inc., Santa Cruz, Calif.), anti-tyrosine hydroxylase
(1:5000; Chemicon), or anti-p75.sup.NTR (1:10,000; Promega). The
membranes were washed four times with TBST (10 minutes each wash),
and then incubated with secondary antibody (1:10,000 goat
anti-rabbit HRP, Boehringer Mannheim, Laval, Quebec, Canada) for
1.5 hours. After three washes in PBST, detection was carried out
using enhanced chemiluminescence (Amersham Canada Ltd., Oakville,
Ontario, Canada) and XAR X-ray film (Eastman Kodak Co., Rochester,
N.Y.).
EXAMPLE 2
BDNF-mediated Activation of p75.sup.NTR Inhibits NGF-induced Growth
of Cultured Sympathetic Neurons
[0133] BDNF-mediated activation of p75.sup.NTR antagonizes
TrkA-mediated sympathetic neuronal survival when NGF levels are
suboptimal, but has no effect on survival at higher levels of NGF.
To determine whether p75.sup.NTR activation also antagonized other
TrkA-mediated biological responses, we focused on sympathetic
neuronal growth. Specifically, we cultured sympathetic neurons in
10 ng/ml NGF and then activated p75.sup.NTR using BDNF. For rat
sympathetic neurons, 10 ng/ml NGF mediates 100% sympathetic neuron
survival but elicits limited morphological growth and TrkA
activation relative to higher concentrations of NGF, while 100
ng/ml BDNF is sufficient to activate p75.sup.NTR in apoptosis
experiments, but does not bind to the two Trk receptors present on
sympathetic neurons, TrkA and TrkC.
[0134] Initially, we observed that the addition of 100 ng/ml BDNF
in the presence of 10 ng/ml NGF had no negative effects on
sympathetic neuron survival (FIG. 1A). Specifically, sympathetic
neurons were cultured for five days in 50 ng/ml NGF, were free of
neurotrophin, and then switched into 10 ng/ml NGF plus or minus 100
ng/ml BDNF. Two days later, neuronal survival was measured using
MTT assays, which measure mitochondrial function. As previously
shown, the addition of 100 ng/ml BDNF had no effect on sympathetic
neuron survival in 10 ng/ml NGF (FIG. 1A).
[0135] We next determined whether p75.sup.NTR activation affected
neuronal growth by measuring the level of neurite extension that
occurs in response to 10 ng/ml NGF with or without BDNF. For these
experiments, neurons were cultured for two to three days in 50
ng/ml NGF, were switched to 10 ng/ml NGF plus or minus 100 ng/ml
BDNF, and the density of neuritic processes was then determined two
days later (FIGS. 1B, 1C). Results from six separate experiments
indicated that p75.sup.NTR activation reduced the process network
density from 22 to 52%, for an average decrease of 40% (FIGS. 1B,
1C, 2A, 2B).
EXAMPLE 3
Autocrine BDNF, Acting through p75.sup.NTR Decreases Growth of
Cultured Sympathetic Neurons
[0136] The foregoing data suggested that exogenous BDNF was able to
activate p75.sup.NTR and negatively-influence TrkA-mediated
neuritogenesis. Since sympathetic neurons themselves synthesize
BDNF that can be detected in conditioned medium obtained from
cultured SCG neurons, we hypothesized that autocrine BDNF might
play a role in negatively regulating levels of sympathetic neuron
growth through an autocrine loop. To test this hypothesis, cultures
were maintained for two to three days in 50 ng/ml NGF, and then
cultured for two more days in 10 ng/ml NGF, plus or minus a
function-blocking anti-BDNF antibody. As a control, we used a
nonimmune control chicken IgY.
[0137] To ensure that the anti-BDNF was capable of neutralizing
BDNF, we incubated TrkB-expressing NIH-3T3 cells for five minutes
with 50 ng/ml BDNF plus or minus 5-40 .mu.g/ml of anti-BDNF. TrkB
protein was then immunoprecipitated using anti-panTrk, and the
immunoprecipitates analyzed by immunoblot analysis with
anti-phosphotyrosine. As controls, cells were incubated either with
culture medium, or medium with BDNF plus 40 .mu.g/ml nonimmune IgY.
This analysis revealed that BDNF caused a robust increase in
tyrosine phosphorylation of TrkB (FIG. 3, lanes 1, 2), and
anti-BDNF inhibited BDNF-stimulated TrkB phosphorylation at
concentrations of 10 .mu.g/ml or higher (FIG. 3, lanes 4-7). In
contrast, the control IgY had no effect on BDNF-mediated TrkB
activation (FIG. 3, lanes 2, 3).
[0138] We then used this function blocking anti-BDNF to test the
role of autocrine BDNF in sympathetic neuron growth. Initially, we
determined whether anti-BDNF had any effect on sympathetic neuron
survival in 10 ng/ml NGF. Neurons were cultured for five days in 50
ng/ml NGF, and then were switched to 10 ng/ml NGF with or without
10 .mu.g/ml anti-BDNF. Measurement of neuronal survival using MTT
assays two days later revealed that anti-BDNF had no effect on
sympathetic neuron survival under these conditions (FIG. 4A). We
then determined whether anti-BDNF affected neuronal growth under
these same conditions; neurons were grown in 50 ng/ml NGF for two
to three days, and then switched into 10 ng/ml NGF plus or minus 10
.mu.g/ml anti-BDNF. Measurement of neurite process density revealed
that cultures exposed to anti-BDNF exhibited an average increase in
neuritogenesis of 80%, relative to 10 ng/ml NGF alone (FIGS. 2A,
2C, 4B, 4C). Nonimmune IgY had no effect on NGF-mediated growth
(FIG. 4B), demonstrating the specificity of the effect. Thus,
autocrine BDNF inhibits TrkA-mediated neuritogenesis in vitro.
[0139] Since we observed autocrine BDNF was mediating these effects
via p75.sup.NTR, we predicted a similar increase in TrkA-mediated
neuritogenesis if we blocked p75.sup.NTR. To test this prediction,
we performed neuritogenesis experiments using the function-blocking
p75.sup.NTR antibody, REX. As before, cultures were initially grown
for two days in 50 ng/ml NGF and were then incubated for two
additional days with 10 ng/ml NGF with or without REX (FIGS. 2A,
2D, 4E, 4F). As a control, sister cultures were incubated with
rabbit serum at the same volume as the REX antiserum. These
experiments revealed that, when p75.sup.NTR was blocked by REX,
neuronal growth was enhanced almost two-fold relative to NGF alone
(FIGS. 2A, 2D, 4E, 4F), an effect that was not observed with rabbit
nonimmune serum (FIG. 4D), and that is similar to the response
elicited by anti-BDNF (FIGS. 4B, 4C). The increased neuronal growth
observed with both REX and anti-BDNF is similar to the 2 to
2.5-fold increase that occurs when NGF is increased from 10 to 40
ng/ml NGF (FIG. 4G), a treatment that causes increased TrkA
activation, supporting the idea that a BDNF:p75.sup.NTR autocrine
loop antagonizes TrkA-mediated sympathetic neuron growth.
EXAMPLE 4
p75.sup.NTR-/- Sympathetic Neurons Show Enhanced Neuronal Growth In
Response to NGF and Do Not Respond to Exogenous BDNF
[0140] Our data strongly indicate that BDNF acts through
p75.sup.NTR to antagonize Trk-mediated enhanced neuronal growth. To
formally demonstrate the necessity of p75.sup.NTR for BDNF's
effects, we cultured neurons from both p75.sup.NTR-/- and wild type
control mice and repeated the neuronal growth assays. Since mouse
sympathetic neurons are more sensitive to NGF than rat sympathetic
neurons, we initially performed survival assays to determine an
appropriate NGF concentration. Specifically, mouse sympathetic
neurons were maintained for five days in 50 ng/ml NGF, were
switched to concentrations of NGF ranging from 0.1 to 10 ng/ml NGF
for three days, and survival was then measured using MTT assays.
These experiments revealed that 5, 7.5, and 10 ng/ml NGF were all
able to mediate maximal mouse sympathetic neuron survival (FIG.
5A). To ensure that BDNF had no apoptotic effect under these
survival conditions, we performed similar experiments with 7.5 or
10 ng/ml NGF plus or minus 100 ng/ml BDNF. MTT assays revealed that
BDNF did not affect mouse sympathetic neuron survival in the
presence of optimal concentration of NGF (FIG. 5B). On the basis of
these data, we selected 7.5 ng/ml NGF for our experiments, a
concentration that was optimal for survival and where BDNF had no
significant effect on survival. We then examined sympathetic
neurons from p75.sup.NTR-/- mice to determine first, whether the
lack of p75.sup.NTR imparted to p75.sup.NTR-/- neurons an intrinsic
ability to extend more neuritic processes, and second, whether BDNF
was acting through p75.sup.NTR to inhibit TrkA-mediated growth. To
perform these experiments, sympathetic neurons were cultured from
p75.sup.NTR-/- versus control mice on the same day, were maintained
for three days in 50 ng/ml NGF, and were subsequently switched to
7.5 ng/ml NGF, plus or minus 100 ng/ml BDNF. Measurement of
neuritic process density revealed that p75.sup.NTR-/- neurons
exhibited an almost two-fold increase in neuronal growth relative
to their wild type counterparts (FIGS. 5C, 5D, 6A, 6C), a result
similar to that observed with the REX and anti-BDNF antibodies in
experiments using rat sympathetic neuron cultures (FIGS. 4B-4E).
Moreover, while BDNF decreased the degree of neuronal growth in
wild type mouse cultures by an average of 35%, exogenous BDNF had
no effect on growth of p75.sup.NTR-/- neurons (FIGS. 5B, 5C, 6C,
6D). Thus p75.sup.NTR is required for BDNF to inhibit NGF-mediated
sympathetic neuronal growth, and NGF is more effective at eliciting
growth in the absence of p75.sup.NTR.
EXAMPLE 5
BDNF is Present in Sympathetic Target Organs and p75.sup.NTR in
Sympathetic Neuron Axons During the Period of Target
Innervation
[0141] Our culture results indicate that BDNF would be present in
sympathetic neuron targets during the developmental period of
target competition. To test this experimentally, we focused on the
pineal gland, a sympathetic target organ that (i) is bilaterally
innervated by neurons from the SCG, (ii) does not receive any other
peripheral innervation from sensory or motor neurons, and (iii) is
innervated postnatally. Ingrowth of sympathetic fibers to the
pineal gland begins during the first week of postnatal life,
reaching adult levels after 3-4 weeks. Lysates of pineal glands
from adult rats were separated by polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and probed for the
presence of BDNF. This analysis revealed a BDNF-immunoreactive band
in the pineal gland that is the same size as BDNF in the rat
cortex, and human recombinant BDNF (FIG. 7A). To confirm the
identity of this band, we analyzed the pineal gland from mice in
which the BDNF gene was mutated by homologous recombination.
Immunoblot analysis revealed that the BDNF-immunoreactive band was
decreased in the pineal glands of P13 to P15 BDNF.sup.+/- mice, and
was completely lost in the pineal glands of BDNF.sup.-/- mice (FIG.
7B). Thus, BDNF is present in the pineal gland at the time of
sympathetic target competition.
[0142] We next determined whether incoming sympathetic axons were
positive for p75.sup.NTR over this same time frame.
Immunohistochemical analysis of the rat pineal gland using the
anti-p75.sup.NTR antibody MC192 revealed the presence of numerous
p75.sup.NTR positive fibers throughout the pineal gland at P6 (FIG.
8J), in a pattern similar to that previously observed for tyrosine
hydroxylase-positive sympathetic fibers. This pattern was somewhat
distinct from that observed in the adult pineal gland, in which
p75.sup.NTR immunostaining is more punctate in nature, reflecting
the mature pattern of sympathetic fibers in the pineal gland (FIG.
8I), as observed for TH-positive sympathetic fibers. To confirm
that this immunostaining corresponded to p75.sup.NTR, we also
performed immunoblot analysis, which demonstrated that p75.sup.NTR
was present in the pineal gland during the first few postnatal
weeks (FIG. 7C). Thus, both BDNF and p75.sup.NTR are present in the
pineal gland at the time of sympathetic target competition.
EXAMPLE 6
The Pineal Gland is Hyperinnervated in BDNF.sup.-/- Mice
[0143] Together, our in vivo and in vitro data predict that, in the
absence of BDNF, sympathetic neuron target innervation should be
increased. To test this experimentally, we examined the level of
sympathetic innervation to the pineal gland of BDNF.sup.-/- mice at
P13 to P15. Initially, we analyzed the density of sympathetic
fibers using immunohistochemistry for tyrosine hydroxylase, which
is specific for sympathetic axons. This analysis revealed that the
plexus of TH-positive fibers was much more dense in the pineal
gland of BDNF.sup.-/- mice than in the pineal gland of control
litter mates (FIGS. 8A-8H). In particular, thick, TH-positive
fibers were interspersed throughout the entire tissue in
BDNF.sup.-/- mice, whereas fibers in the pineal gland of control
litter mates were less abundant and appeared qualitatively
thinner.
[0144] To confirm this increase in TH-positive fibers, we also
measured the level of TH in the pineal gland biochemically at P13
to P15. Western blot analysis revealed that TH levels were
significantly increased in the pineal gland of BDNF.sup.-/- animals
relative to their control litter mates (FIG. 7D). TH levels were
similarly increased in the pineal gland of BDNF.sup.+/- litter
mates (FIG. 7D), coincident with decreased BDNF (FIG. 7B),
indicating that the increased sympathetic innervation was not due
to any potential developmental delay in the BDNF.sup.-/- mice.
Moreover, p75.sup.NTR levels were similar in the pineal glands
regardless of the BDNF gene dosage (FIG. 7E), indicating that the
differences in levels of innervation are not due to differences in
levels of this receptor. Thus, when BDNF is either reduced or
absent, both the density of sympathetic innervation and levels of
TH are increased.
EXAMPLE 7
Genetic Perturbation of NGF-Mediated Neuronal Growth
[0145] We have presently discovered that MEK/MAPK signaling
modulates neuronal growth, without modulating cell survival.
Accordingly, a compound that blocks MEK/MAPK signaling would block
neuronal growth without affecting cell survival. One mechanism for
blocking or inhibiting MEK/MAPK signaling is to use a
dominant-negative MEK protein, such as DN MEK (FIG. 9A), in which
lysine 97 has been replaced with a methionine (see, for example
Holt et al., Mol. Cell Biol. 16:577-583, 1996). Using such an
approach we demonstrated that MEK/MAPK signaling is required for
neuronal growth both in vitro and in vivo.
[0146] We first inhibited MEK activity in vitro using a recombinant
adenovirus expressing DN MEK. We also examined the role of MEK in
vivo by generating transgenic mice which express DN MEK under the
control of a neuron-specific and pan-neuronal T.alpha.1 .alpha.
tubulin promoter (T.alpha.1: DN MEK).
[0147] Nucleic acid molecules encoding one of three HA-tagged MEK
were cloned into an adenoviral vector which expresses green
fluorescent protein (GFP) in a dicistronic fashion. WT MEK encoded
wild-type MEK, DN MEK, in which lysine 97 was replaced with a
methionine, encoded a dominant negative MEK, and ACT MEK, in which
serine 218 and serine 222 were replaced by aspartic acid and
glutamic acid, respectively, encoded an activated MEK (FIG.
9A).
EXAMPLE 8
DN MEK-Infected Cells Show Decreased MAPK Activation Following
Treatment with NGF
[0148] Within 10 minutes of treatment with NGF, PC12 cells show
marked increase in the phosphorylation of MAPK; prior infection of
PC12 cells with an adenovirus encoding WT MEK did not diminish this
phosphorylation event (FIG. 9B). In contrast, prior infection of
PC12 cells with DN MEK resulted in a dramatic decrease in MAPK
phosphorylation, as determined by western blotting with an antibody
that specifically recognizes phosphorylated MAPK (P-MAPK), and not
unphosphorylated MAPK (FIG. 9B). This decrease in P-MAPK is not due
to an overall decrease in protein, as western blots of WT MEK and
DN MEK-infected cells with anti-HA revealed the same amount of
HA-tagged protein.
[0149] Neurons isolated from rat superior cervical ganglia were
also infected with either WT MEK or DN MEK. As demonstrated for the
PC12 cells, DN MEK decreased levels of MAPK phosphorylation (FIGS.
9B, 9C).
EXAMPLE 9
Survival of SCG Neurons in the Absence of NGF is not Enhanced
Following Infection with MEK-Encoding Adenovirus
[0150] One explanation for the observed decrease in P-MAPK levels
in SCG neurons was that expression of various MEK constructs
altered cell survival. We performed a survival assay designed to
detect survival-altering activity of MEK after NGF withdrawal. None
of the constructs showed a significant alteration in cell survival
(FIG. 9D). These data indicate that DN MEK reduced MAPK
phosphorylation without altering cell survival.
EXAMPLE 10
DN MEK Decreases Neuronal Growth
[0151] We have devised a method for quantitatively assaying neurite
growth. In this assay, the cell bodies of neurons are plated in
Campenot chamber, a compartmentalized dish that has scratches or
grooves in the matrix. The compartments are physically separated
from each other by, for example, a Teflon.RTM. divider. The cells
are placed in the central compartment. The scratches allow neurites
to extend from the neuronal cell bodies and into adjacent
compartments (FIG. 10A).
[0152] Neurites were induced to grow into HGF supplemented side
compartments, and then visualized by staining against tubulin.
Adenoviral-mediated expression of DN MEK in SCG neurons decreased
neuritogenesis by 39% (FIGS. 10B, 10C).
EXAMPLE 11
Generation and Phenotvpe of Transgenic Mice that Express DN MEK
[0153] Using standard techniques, we generated five lines of
transgenic mice that express DN MEK under the control of the
T.alpha.1 .alpha. tubulin promoter. FIG. 11 shows a
beta-galactosidase-stained embryo at E13.5 of T.alpha.1: lacZ
reporter mouse, demonstrating that the T.alpha.1 .alpha. tubulin
promoter is targeted and limited to the central and peripheral
nervous systems. Peak of expression was found to be at E16.5, with
declining levels thereafter.
[0154] In order to determine whether DN MEK decreased P-MAPK
levels, we analyzed proteins from tissue lysates of hippocampi
dissected from transgenic P4 mice and nontransgenic controls.
Densitometric quantification showed that transgenic expression of
DN-MEK reduces P-MAPK reactivity in the hippocampus to
approximately 50% of the wild-type value (FIG. 12).
[0155] We examined the sympathetic innervation of pineal glands by
immunohistochemistry against tyrosine hydroxylase (TH). Transgenic
liter mates showed a significant loss of sympathetic innervation
compared to non-transgenic controls (FIG. 13A). Computer-based
imaging was used to quantify innervation. Using this method, we
determined that sympathetic innervation of the pineal gland was
reduced by approximately 75%. Loss of sympathetic innervation of
the pineal gland is not due to cell death. By counting sympathetic
neurons of the SCG, we found no difference between wild-type and
transgenic litter mates. We conclude that loss of pineal gland
innervation in DN MEK transgenic animals is not due to increased
cell death of SCG neurons (FIG. 13B).
[0156] T.alpha.1 :DN MEK mice also have a spatial loss of
unmyelinated C-fibers in the medial branch of the dorsal cutaneous
artery. Counting of axons revealed a loss of approximately 35% of
unmyelinated axons (C-fibers) (FIGS. 14A, 14B), while the number of
myelinated axons remain constant.
EXAMPLE 12
Pharmacologic Inhibition of MEK Activity Blocks Trk-Mediated
Neuronal Growth in Primary Neurons
[0157] We cultured postnatal sympathetic neurons in Campenot
chambers. Cells exhibit robust neuronal growth into a side
compartment containing 10 ng/ml NGF. Addition of 50 .mu.M U0126 in
conjunction with 10 ng/ml NGF abrogates this growth while having no
effects on cell survival (FIG. 15).
[0158] MEK activity is also required to transduce TrkB-mediated
neuronal growth. BDNF does not induce neuronal growth of
sympathetic neurons because the neurons lack TrkB receptors. We
transduced postnatal sympathetic neurons with an adenovirus
encoding TrkB. In Campenot dishes, these neurons grow processes
into compartments containing 25 ng/ml BDNF. Similar to the effects
on Trk-mediated growth, this TrkB-mediated neuronal growth is
dramatically reduced in a dose-dependent manner when the MEK
inhibitor U0126 is added in conjunction with BDNF (FIG. 16).
[0159] We confirmed the requirement of MEK signaling for
TrkB-mediated neuronal growth by coinfecting two adenoviruses,
expressing DN MEK and TrkB, respectively. Neurons expressing both
constructs exhibited greatly diminished neuronal growth in response
to 25 ng/ml BDNF, as compared to neuronal growth mediated by TrkB
expression alone.
EXAMPLE 13
A mutant TrkB Receptor Exhibiting Reduced Activation of MEK is
Severely Deficient in Promoting Neuronal Growth
[0160] An advantage of using BDNF and exogenously delivered TrkB,
rather than NGF and the endogenous TrkA found in sympathetic
neurons, is that mutated or modified receptors can be introduced
and tested without the complication of the presence of wild-type
receptors. In one example, a TrkB receptor harboring a mutation in
the SHC-association site was introduced in an adenoviral vector.
Following addition of BDNF, this mutated receptor stimulated MEK
only very weakly, as revealed by phosphorylation levels of MAPK
(FIG. 17A) and neuronal growth (FIG. 17B). Thus, using such a
strategy, one can identify mutant forms of TrkB that modulate
neuronal growth without modulating neuronal survival. Such mutant
forms may be useful in gene therapy.
EXAMPLE 14
Activity-Dependent Dendrite Formation in Sympathetic Neurons
Neuronal Activity Causes Reversible Dendrite Formation in
Sympathetic Neurons, and NGF Enhances this Effect
[0161] Neuronal depolarization causes dendrite formation in
cultured sympathetic neurons, and NGF enhances this
dendritogenesis. FIGS. 19A-19C, 20A, 20B show immunocytochemical
analysis of sympathetic neurons cultured in the presence of NGF,
switched to 10 ng/ml NGF, 50 mM KCl, or 10 ng/ml NGF+50 mM KCl for
two days, and then stained for the dendrite-specific protein MAP2.
FIGS. 20A and 20B are high magnification views of fields similar to
those shown at lower-magnification in FIGS. 19A and 19C,
respectively. The MAP2-immunoreactive processes show morphology
characteristic of dendrites. The amount of dendritic formation
could be readily quantified. Image analysis was used to measure the
length of MAP2-positive dendrites on sympathetic neurons cultured
in NGF, KCl, or NGF+KCl, and then these data were plotted as a
distribution histogram (FIG. 21). Note that, while NGF on its own
has little ability to cause dendritic growth, the addition of NGF
to 50 mM KCl greatly enhances the KCl-dependent effect.
[0162] We found that activity-dependent dendrite formation is
reversible. Immunocytochemical analysis of sympathetic neurons
stained for MAP2 after being cultured in the presence of NGF,
switched to 10 ng/ml NGF, 50 mM KCl, or NGF+KCl for three days, and
then maintained in the same conditions or switched back to 10 ng/ml
NGF for an additional two days indicated that, when neurons are
maintained in depolarizing conditions, the MAP2-positive dendrites
retract when KCl is removed (FIGS. 23A-23D).
[0163] Depolarization Promotes the Association of MAP2 with
Microtubules, and Enhances Microtubule Stability
[0164] FIG. 22 shows quantitation of the amount of MAP2 associated
with microtubules in sympathetic neurons switched into 10 ng/ml
NGF, 50 mM KCl, or NGF+KCl for two days. Microtubules were isolated
from cultured sympathetic neurons, and the relative amount of MAP2
present per microgram tubulin was determined using quantitative
dot-blot analysis. Note that KCl greatly enhances the amount of
MAP2 bound to microtubules, and that NGF enhances this effect to
some degree. Depolarization enhances MAP2 levels and CamKII
phosphorylation in sympathetic neurons. We performed western blot
analysis of sympathetic neurons treated with 10 ng/ml NGF, 50 mM
KCl, or NGF+KCl. Lysates containing equal amounts of protein were
separated by SDS-PAGE, transferred to nitrocellulose, and then
probed for MAP2, phosphorylated-CamKII, phosphorylated-ERKs, or
total ERK protein. KCl increases the amount of MAP2 present in
sympathetic neurons, and enhances CamKII phosphorylation. In
contrast, 10 ng/ml NGF is a more potent stimulator of ERK
phosphorylation than is 50 mM KCl.
[0165] Depolarization leads to CamKII phosphorylation in dendrites
of sympathetic neurons. In order to verify that depolarization
results in CamKII phosphorylation, we performed immunocytochemical
analysis of sympathetic neurons switched into 10 ng/ml NGF, or 10
ng/ml NGF+50 mM KCl for two days. Neurons were double-labelled with
antibodies specific to MAP2 and to phosphorylated-CamKII. In the
presence of NGF alone, both MAP2 and phosphorylated-CamKII
immunoreactivity are limited to the cell bodies of the cultured
neurons. In contrast, when cultured in NGF+KCl, MAP2 is localized
to dendrites and cell bodies, and phospho-CamKII immunostaining
colocalizes in those dendrites. In addition, phospho-CamKII
immunoreactivity is detectable in a particulate pattern along the
length of neurites that are not dendrites.
[0166] Activation of Both CamKII and MEK are Essential for
Activity-Dependent Dendritogenesis
[0167] Inhibition of CamKII or ERKs does not affect the survival of
sympathetic neurons in NGF+KCl. We first established that the two
Cam kinase inhibitors used in this study, KN-62 and AIP, do not
affect ERK or Akt activation, that PD98059 does not affect CamKII
activity, and that these drugs do effectively block the appropriate
pathway under our conditions. We then performed MTT survival assays
of sympathetic neurons switched to 10 ng/ml NGF, 50 mM KCl, or NGF+
plus KCl with or without the addition of KN-62 (inhibits CamKII),
U0126 (inhibits MEK), or H-89 and forskolin (both of which perturb
cAMP). None of these compounds perturbs sympathetic neurons
cultured in the presence of NGF or NGF+KCl. In contrast, KN-62
dramatically reduces the survival of sympathetic neurons cultured
only in 50 mM KCl.
[0168] Inhibition of either CamKII or MEK is sufficient to inhibit
activitydependent dendrite formation. Immunocytochemical analysis
for MAP2 on sympathetic neurons cultured in the presence of NGF+KCl
with or without 10 um KN62, 75 um PD98059, 100 nM H-89 or 10 uM
forskolin. Inhibition of either CamKII or MEK completely abolished
activity-dependent dendrite formation (FIGS. 24A-24J). Similar
results were obtained with AIP (a second CamKII inhibitor) and
U0129 (a second MEK inhibitor). These data are quantified in FIG.
25. Inhibition of CamKII or MEK also inhibited
depolarization-induced association of MAP2 with microtubules, and
the coincident increase in microtubule stability (FIG. 26). In this
study, microtubule stability was determined in sympathetic neurons
maintained in NGF or NGF+KCl, and treated with selective
pharmacological inhibitors. Note that both KN62 and U0126
completely inhibit the KCl-induced increase in microtubule
stability.
[0169] Other Embodiments
[0170] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each independent publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0171] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure come within
known or customary practice within the art to which the invention
pertains and may be applied to the essential features hereinbefore
set forth.
* * * * *
References